The present invention relates to an analytical assay procedure for determining the amount of an analyte present in a liquid test sample. In particular, the present invention relates to the determination of an analyte in a liquid test sample involving analytical reactions between the analyte and one or more analytical reagents requiring sequential manipulative steps to make such determination.
In U.S. Pat. No. 4,990,075 there is disclosed a self contained analytical reaction vessel or device and method for performing analytical assay procedures involving sequential analytical reactions between an analyte in a liquid test sample and one or more analytical reagents which interact with the analyte to produce a detectable response as a function of the analyte. The device is described as being particularly useful for performing immunoassays which typically require a number of cumbersome manipulative steps such as pipetting, mixing and incubation of the liquid test sample with the analytical reagents.
More particularly, this device is described as comprising a closed container having a substantially horizontal axis of rotation and an analytical reagent reaction channel, liquid test sample delivery means for facilitating the flow of a liquid test sample into the reaction channel. The analytical reaction channel comprises one or more reaction zones incorporated with one or more analytical reagents in the dry form. A first analytical reagent is incorporated into the first reaction zone and a second reagent or reagents into the second reaction zone which is in liquid communication with the first reaction zone. A liquid test sample disposed in the reaction channel can be transported by gravity along the reaction channel between the reaction zones by rotating the device along its horizontal axis of rotation. This device works well when only one reagent is in the second reaction zone since after dissolution of the first analytical reagent the device is simply tilted to bring the reaction fluid into contact with the second dry reagent in the second reaction zone and allowed to remain in this configuration until the second reagent completely dissolves. However, in certain analytical procedures, there must be two or more dry reagents in the second reaction zone. When these reagents have different rates of dissolution, and are being dissolved simultaneously in the same fluid, certain problems are encountered. For example, if the dissolution rates of the two reagents are slow and the reaction kinetics are fast, most of the reaction will have been completed before all of the reactants are dissolved thereby interfering with the measurement of the initial kinetics of the reaction, which are the most sensitive part thereof.
In U.S. Pat. No. 4,970,171 there is disclosed an analytical method for determining glycosylated hemoglobin wherein the amounts of both total hemoglobin and glycosylated hemoglobin derivative are measured and related as a percentage. In this method, a blood sample is treated with a thiocyanate salt and an oxidant to denature the hemoglobin in the sample thereby converting it to met-hemoglobin. The met-hemoglobin is measured spectrophotometrically to give the total amount of hemoglobin in the sample while the denatured glycosylated hemoglobin can be measured by immunoassay. This patent describes a particle agglutination inhibition assay based on the specific interaction of an antibody particle reagent and an agglutinator. The antibody particle reagent comprises the antibody, or a fragment thereof, bound to a water suspensible particle, e.g. a polystyrene or other latex, and the agglutinator comprises a polymeric material bearing a plurality of epitopic binding sites for the antibody reagent. This sort of immunosassay format is well known to those skilled in this art. The above described assay for glycosylated hemoglobin is well suited for adaptation to the reaction vessel for performing sequential analytical assays. Thus, placing the dry oxidant/isothiocyanate in the first reaction zone and dissolving it in the reaction fluid containing a blood sample to thereby cause the denaturation of the blood's hemoglobin and rotating the vessel to cause the reaction fluid to come into contact with dried antibody bearing latex and agglutinator as the second reagents in the second reaction zone, facilitates the immunoagglutination assay upon dissolution of the latex and agglutinator. However, since the latex and agglutinator go into suspension at different rates, a problem can be encountered since the latex reagent dissolves much more slowly than the agglutinator, the majority of the reaction occurs before all of the reactants are dissolved. Accordingly, the initial agglutination reaction, which is the most sensitive part of the assay, cannot be measured with the maximum degree of accuracy attainable for this type of immunoassay.
It is an object of the present invention to provide a method and device which are useful for carrying out the above described sequential assay while obviating the problems associated with the dissolution of two or more analytical reagents in the second reaction zone.
The present invention is described in greater detail in the following discussion. Since the latex reagent does not form a true solution with the reaction fluid, the terms dissolution and solution as used herein are intended to include the colloidal suspension of the latex particles having antibody bound thereto as well as true solutions.